The goal of the proposed project is to develop cell-based assays to analyze neurotransmitter release at the human dopamine, norepinephrine, and serotonin biogenic amine transporters (BATs). Both types of BAT ligands (reuptake inhibitors and substrate-type releasers) elevate extracellular neurotransmitter concentration, but reuptake inhibitors bind to transporters, while substrate-type releasers are transported inside the neuron and induce neurotransmitter release. Since many psychostimulants and designer drugs such as bath salts interact with BATs, understanding the mechanism of this interaction proves very useful in determining how to better treat addiction disorders. Currently, the typical assays for assessing the BAT activity of general compounds involve using fresh rat brain synaptosomes or transfected cells, both of which have unresolved issues. While the rat brain synaptosome has native transport machinery and distinguishes between release and reuptake inhibition in expected potency ranges based on human use, the assay does not measure human transporter activity. The use of rat synaptosomes also lowers efficiency, increases the cost, prevents high-throughput screening of transporter activity, and raises ethical concerns due to the use of animals. Current cell-based assays use generic cell lines that lack native transport machinery and contain transfected human transporters; both characteristics lead to the inability to distinguish between BAT releasers and reuptake inhibitors. Therefore, there is a need to improve upon the current methods in order to produce better human transporter neurotransmitter release data while lowering cost, removing ethical concerns, and increasing overall assay efficiency. The proposed project will investigate cell lines with endogenous human transporters and native transport machinery required for transporter-mediated release. Following evaluation of the neurotransmitter release and uptake activity of the cell lines, ligands with known activity at rat BATs will be tested. Results from the human cells will be compared with rat brain synaptosome data in order to determine how the ligand potencies compare between models and how well the human cells discriminate between release and reuptake inhibition. The ligands will also be analyzed in HEK293 cells with over-expressed transporters in order to compare endogenous transporter activity with transfected transporter activity.